Immunohistochemical Expression of Mast Cell in Oral Reactive Lesions

Statement of the Problem Soft tissue reactive lesions are the most common lesions of the oral cavity. Although many studies have shown the interaction of mast cells with fibroblasts and their participation in fibrosis, the role of mast cells in these lesions is not well understood. Purpose The aim of this study was to evaluate the mast cells (MCs) count in oral soft-tissue reactive lesions including peripheral giant cell granuloma (PGCG), peripheral ossifying fibroma (POF), irritation fibroma (IF) and normal oral mucosa. Materials and Method In this cross-sectional study, 50 samples including IF, PGCG, POF (14 cases for each group) and 8 cases of normal oral mucosa were stained with tryptase antibody through immunohistochemistry. The number of mast cells was counted in 5HPF containing maximum counts for each section stained with tryptase. Statistical analysis including Chi-square test and Tukey test with a significance level of 0.05 were considered. Results The number of MCs was found to have increased in reactive lesions compared with normal oral mucosa. MCs count in the POF group was higher than the others. Conclusion These findings suggest a possible role of mast cells in the pathogenesis of reactive oral lesions and induction of fibrous tissues. Chemical mediators released from mast cells might influence other cells, especially fibroblasts, to induce fibrosis.


Introduction
Soft tissue lesions of the oral cavity comprise a wide variety of lesions from reactive conditions to neoplasms.
Reactive lesions of the oral soft tissues are the most common lesions that occur in the oral cavity and are derived from mesenchymal cells. [1] Considering their histopathological features, they may present fibrotic hyperplasia like in irritation fibroma or may show proliferation of granulation tissue like in pyogenic granu-lomas. [1] These lesions are developed in response to local irritations. [2] The most common reactive lesions of soft tissue are irritation fibroma, peripheral giant cell granuloma (PGCG), and peripheral ossifying fibroma (POF). Fibroma is the most prevalent tumor of the oral cavity. [2] There is still controversy whether fibroma is a real neoplasm or a reactive hyperplasia of the connective tissue in response to trauma and local irritations.
Microscopic evaluation of irritation fibroma reveals nodular masses of collagenized and dense fibrous connective tissue. [2] PGCG is a relatively frequent tumorlike lesion of the oral cavity which is developed due to trauma or local irritation. Microscopic examination of PGCG exhibits a large number of multi-nucleated giant cells, in addition to proliferation of fibroblasts. [2] POF is a relatively common gingival hyperplasia that is composed of proliferation of fibroblasts along with mineralized components. These lesions are treated by conservative surgical excision. [2] Mast cells are components of the immune system, which originate from bone marrow and are found in all the connective and mucous tissues, especially around vessels, and in the peripheral and central nervous systems. They are round to oval in shape and are one-nucleated in histologic sections, with a large number of granules in the cytoplasm. [3][4] The primary role of these cells is to take part in hypersensitivity reactions and inflammatory processes. Evidence shows the role of mast cells in the formation of matrices, granulation tissue, wound healing and angiogenesis. [3][4] On the other hand, the study has shown interaction of mast cells with fibroblasts and their relation with the synthesis of collagen in a large number of pathologic lesions such as scleroderma, submucous fibrosis of the oral cavity, gingival fibromatosis and fibrotic changes in the salivary glands of patients with Sjögren's syndrome. [3] Several techniques are available to identify mast cells in different lesions, one of which is the use of immunohistochemical markers. In the present study, tryptase was used which is the most common serine protease of secretory granules of mast cells; it is an appropriate marker to identify these cells. [5][6] Since only a limited number of studies have been carried out on the role of mast cells in the pathogenesis of reactive lesions of soft tissues of the oral cavity [3,7] and because reactive lesions are the most common lesions of oral cavity, the present study was undertaken to identify mast cell in such lesions, by using immunohistochemistry (IHC).

Materials and Method
In this cross-sectional descriptive/analytical study, the Two oral pathologists evaluated stained slides under a light microscope (Olympus BX41; Japan) at ×100 and ×400 magnifications. Five hot spot fields were selected under low magnification (×100) and mast cells were counted in each at ×400 magnification and the mean number of mast cells in the five regions was obtained. [8] Data analysis was done using SPSS software (ver.16). The variance and Tukey test were used. Statistical significance was defined at p≤ 0.05.

Results
Of all the samples evaluated, 22 belonged to men (44%) and 28 to women (56%). Out of all the PGCGs, 57.1% belonged to men; the samples of POFs and normal mucosa equally belonged to men and women (50%); and of all the irritation fibroma samples, 78.6% belonged to women.
The mean age of the subjects was 34±16.9 years, with a range of 7-76 years. The most and least commonly involved areas were gingiva (48%) and the tongue (2%), respectively. The mean tryptase-positive mast cell count was 28.1±10.4 in POF, 23.5±7.9 in PGCG, 18.9±8.1 in irritation fibroma, and 13.7±4.2 in normal oral mucosa (Figure 1). Using variance analysis, statistically significant differences were obtained in mast cell counts between the study groups (p= 0.002, F=6), with the highest counts in POF (Table 1). Tukey tests showed statistically significant differences in mast cell counts among POF, irritation fibroma, and normal oral mucosa, (p= 0.005), (p= 0.002) respectively. However, there was no significant difference between POF and PGCG (p=0.15). In addition, there were no significant differences among irritation fibroma, normal oral mucosa (p= 0.17), and PGCG (p= 0.16).  Akers et al. [14] and Asano-Kato et al. [15] used the culture technique in their studies and showed that tryptase with protease activated receptor-2 resulted in proliferation of fibroblasts in the conjunctiva. In a study, Garbuzenko et al. revealed that human mast cells have a role in skin remodeling and fibrosis [13].

Discussion
In a rather similar study, Shahrabi et al. which showed statistically significant differences only between POF and normal mucosa, in that study there were significant differences between all the groups and normal gingiva regarding the number of mast cells. [3] The differences in these results might be attributed to the differences in the techniques used in these two stud- gland, especially in glands with more fibrous sialoadenitis, and the difference was statistically significant. [11] The results of that study were consistent with those of the current one considering the increase in mast cell counts in fibrous tissues compared with normal samples.
In the studies by Andersson et al. [12] and Sabar-